Page 1 of 24
Persistent organic pollutants and
1
organophosphate esters in feathers and
2
blood plasma of adult kittiwakes (Rissa
3
tridactyla) from Svalbard – associations
4
with body condition and thyroid hormones
5 6 7
N. B. Svendsen1*, D. Herzke2, M. Harju2, C. Bech1, G. W. Gabrielsen3, V. L. B. Jaspers1 8
9
1 Department of Biology, Norwegian University of Science and Technology (NTNU), NO-7491 10
Trondheim, Norway 11
2 Norwegian Institute for Air Research (NILU), FRAM Centre, NO-9296 Tromsø, Norway 12
3 Norwegian Polar Institute, FRAM Centre, NO-9296 Tromsø, Norway 13
*Corresponding author: [email protected], +45 6039 4410 14
15
Page 2 of 24 Abstract
16
Polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine 17
pesticides (OCPs) and organophosphate esters (OPEs) were assessed in blood plasma and 18
feathers of 19 adult black-legged kittiwakes (Rissa tridactyla) breeding in two colonies 19
(Blomstrandhalvøya and Krykkjefjellet) at the Arctic archipelago, Svalbard. Potential 20
associations with body condition index (BCI) and thyroid hormones were investigated. All 21
compound classes were detected in both blood plasma and feathers, but due to low sample 22
size and volumes, OPEs could only be quantified in four individuals, warranting larger follow 23
up studies. Kittiwakes breeding at Blomstrandhalvøya had significantly higher concentrations 24
of organic pollutants in blood plasma than kittiwakes breeding at Krykkjefjellet (p < 0.001).
25
Concentrations in blood plasma and feathers did not significantly correlate for any of the 26
investigated compounds, and feather concentrations did not differ significantly between the 27
colonies. This suggests that pollutant levels in adult kittiwake feathers do not reflect local 28
contamination at breeding sites and are as such not useful to monitor local contamination at 29
Svalbard. Significant negative associations between BCI and most pollutants were found in 30
both populations, whereas significant correlations between the BCI, the ratio of total 31
triiodothyronine to free triiodothyronine (TT3:fT3), and several pollutants were only found for 32
kittiwakes from Blomstrandhalvøya (all r ≥ -0.60 and p ≤ 0.05). This indicates that higher 33
levels of circulating pollutants during the breeding period covary with the TT3:fT3 ratio, and 34
may act as an additional stressor during this period.
35 36
Keywords: Feathers, POPs, organophosphate esters, thyroid hormones, black-legged 37
kittiwakes 38
39
Page 3 of 24 Funding sources
40
N. B. Svendsen and V. L. B. Jaspers received Artic Field Grants (ES520962 and ES520958, 41
respectively) from the Norwegian Research Council to carry out the fieldwork at Svalbard, 42
Norway. V.L.B Jaspers is further supported by the Norwegian University of Science and 43
Technology (NTNU), and the pollutant analyses were funded in a joint collaboration between 44
NTNU and the Norwegian Polar Institute.
45 46
Page 4 of 24 1. Introduction
47
The first reports of contaminated Arctic wildlife were published in the early 1970’s (AMAP 48
1998), and now the Arctic is considered as an important indicator region for assessing the 49
persistence and bioaccumulative abilities of emerging contaminants (de Wit et al. 2010).
50
Atmospheric transport is the main and most rapid source of semi-volatile persistent organic 51
pollutants (POPs) to the Arctic (Gordeev 2002; AMAP 2015). In the Arctic, POPs enter 52
seabird species, such as the black-legged kittiwake (Rissa tridactyla, hereafter just 53
‘kittiwake’), mainly through their diet, and are thereafter distributed to lipid rich tissues (AMAP 54
2015). During the reproductive period, when seabirds are believed to function close to their 55
physiological limit (Bech et al. 2002), they rely on energy stored as lipids. Therefore, mass 56
loss during the breeding period is common in birds (Moreno 1989) and kittiwakes are no 57
exception (Henriksen et al. 1996; Bech et al. 2002). This release of lipids to the blood leads to 58
a redistribution of lipophilic contaminants, which increases the concentration of circulating 59
pollutants, and the risk that POPs can reach sites of toxicity (Henriksen et al. 1996). Hence, 60
during the breeding period kittiwakes may be at higher risk of negative effects associated with 61
POPs, than the mean concentration of POPs might suggest (Macdonald and Brewers 1996).
62
In Arctic seabird species, several effects have already been related to POP exposure. These 63
include changed reproductive behavior, reduced adult survival rate, wing feather asymmetry, 64
suppressed immune function, reduced offspring performance, and lowered levels of 65
circulating thyroid hormones (THs) (Grasman et al. 1996; Bustnes et al. 2001; Bustnes et al.
66
2003; Verreault et al. 2004; Verboven et al. 2009; Nøst et al. 2012). In the present study, all 67
investigated legacy POPs, including organochlorine pesticides (OCPs), polybrominated 68
diphenylethers (PBDEs) and polychlorinated biphenyls (PCBs) have the potential properties 69
to be endocrine disrupting chemicals (EDC; Petersen et al. 2007). EDCs may have adverse 70
effects on the TH system, which is vital for seabirds to adapt, reproduce, and survive in the 71
cold Arctic climate (Gabrielsen 2007).
72
In birds, the predominant TH is thyroxine (T4), whereas the biologically active TH is 73
triiodothyronine (T3) (McNabb 1995). T4 is transported in blood mainly by the transport 74
proteins transthyretin and albumin (McNabb 2007; Hill et al. 2008), and mostly converted to 75
Page 5 of 24 the active form T3 by hepatic type 1 deiodinase (Dawson 2000). Active THs exert a wide 76
range of effects and are required for growth, differentiation and maturation of several body 77
systems, central nervous system development, and reproductive activity (Dawson, 2000;
78
McNabb 2007). THs also induce molt and regulate heat production in order to maintain a 79
constant body temperature, which is crucial for Arctic seabirds (McNabb 2007). Since the 80
Arctic summer is short, proper timing of breeding, molting, and migration is essential for 81
survival. Exposure to EDCs could disrupt the ability of the endocrine system to regulate these 82
events as some EDCs have structural resemblance with THs (Verreault et al. 2004) and may 83
cause decreased T3 levels (Blévin et al. 2017). This could lead to less successful breeding 84
and in the worst case reduced survival (Jenssen 2006).
85
Studies, that have investigated the use of feathers for measuring POPs and emerging 86
pollutants, have evaluated feathers as a useful biomonitoring tool for non-destructive 87
detection and quantification of organic pollutants (Dauwe et al. 2005; Jaspers et al. 2006;
88
Jaspers et al. 2007b; van den Steen et al. 2007; Eulaers et al. 2011; García-Fernández et al.
89
2013). (Re-)emerging pollutants, such as organophosphate esters (OPEs), have been 90
detected in the Arctic environment (Salamova et al. 2014), but very few studies have 91
investigated their occurrence in Arctic wildlife (Evenset et al. 2009; Hallanger et al. 2015). The 92
present study further addresses this issue by examining POPs and OPEs in feather and blood 93
samples from kittiwakes breeding at the Arctic archipelago, Svalbard.
94
The main objectives of the present study were to 1) assess plasma and feather 95
concentrations of PCBs, OCPs, PBDEs, and OPEs; 2) examine the relationship between 96
pollutant levels in feathers and blood; 3) evaluate potential correlations between pollutants 97
and thyroid hormones in kittiwakes breeding at Svalbard.
98 99
Page 6 of 24 2. Materials and methods
100
2.1 Study area and sample 101
collection 102
Sampling was conducted during the 103
kittiwake breeding season in July and 104
August 2014. Two colonies located 105
close to Ny-Ålesund, Kongsfjorden, 106
Svalbard (78°55’N, 11°55’E), Norway, 107
were studied – the ‘Krykkjefjellet’
108
colony approximately 7 km southeast 109
of Ny-Ålesund, and the 110
‘Blomstrandhalvøya’ colony on the 111
northeast side of Blomstrandhalvøya 112
(Fig. 1). Eight birds (5 males, 3 113
females) from Krykkjefjellet were 114
sampled mid-July to early-August, and 115
eleven birds (6 males, 5 females) from 116
Blomstrandhalvøya were sampled in 117
early-August. All sampled kittiwakes 118
were adult and caught on their nest or adjacent cliffs with a noose at the end of a 5 m long 119
fishing rod. Biometric measurements of weight, skull-, tarsus- and wing length, as well as 120
blood and feather sampling were carried out immediately after capture. Feathers from the 121
back, the head, and the sixth primary feather (both wings) were sampled and pooled for 122
analysis. Approximately 2 mL of blood was drawn from the alar vein with a 2 mL heparinized 123
syringe (25 G) and stored on ice until samples were centrifuged at 4000 rpm and then frozen 124
(-20 °C) until analysis. All handling and sampling of the birds occurred by trained personnel 125
Figure 1. An overview of Kongsfjorden situated on the west side of the Arctic archipelago Svalbard, Norway. The two colonies are marked with an asterisk. All map data are from the Norwegian Polar Institute. Map design: Niels Borup Svendsen.
Page 7 of 24 and was in accordance with ethical guidelines and approval by the Norwegian Animal
126
Research Authority (FDU permission number 2014/59453-2).
127
2.2 Sex determination 128
All birds were sexed at the Norwegian University of Science and Technology (NTNU) in 129
Trondheim, Norway, following methods described by Griffiths et al. (1998). In short, DNA was 130
isolated from blood samples by using the Chelex method as described by Walsh et al. (1991), 131
and Chromobox-helicase-DNA-binding genes (CHD-W and CHD-Z) were amplified by PCR.
132
The avian sex chromosome CHD is widely used for sexing purposes, and as CHD-W only 133
occurs in females (ZW) and not in males (ZZ), PCR products separated by electrophoresis 134
result in one band for males and two bands for females.
135
2.3 Thyroid hormone analysis 136
Total triiodothyronine (TT3) and free triiodothyronine (fT3) were quantified in plasma by a 137
competitive enzyme immunoassay human kit (MP Biomedicals, Ohio, USA) at NTNU, 138
Trondheim. Two blank samples and a human T3 standard reference set were used as quality 139
assurance of the quantification. The mean of two replicates was calculated for both TT3 and 140
fT3 with an average intra-assay coefficient of variation (CV) of 10 % for fT3 and 6 % for TT3.
141
Levels of T4 and glandular hormones could not be investigated due to limited plasma 142
amounts.
143
2.4 Contaminant analysis 144
Contaminant analyses were conducted at the Norwegian Institute for Air Research (NILU) in 145
Tromsø, Norway. In all samples, 8 PBDE congeners (28, 47, 99, 100, 138, 153, 154 and 146
184), 12 PCB congeners (28, 52, 99, 101, 105, 118, 138, 153, 180, 183, 187 and 194), 147
hexachlorobenzene (HCB), oxy-, cis- and trans-chlordane (OxC, CC, and TC), cis- and trans- 148
nonachlor (CN and TN), mirex, α-, β-, and γ-hexachlorocyclohexane (HCH), o,p’-DDT and 149
p,p’-DDT and transformation products (p,p’-DDD, o,p’-DDD, p,p’-DDE and o,p’-DDE) were 150
analyzed. In four individuals, the following 13 organophosphate esters were analyzed in both 151
feathers and blood as well: tris(2-chloroethyl) phosphate (TCEP), tripropyl phosphate (TnPP), 152
Page 8 of 24 tris(2-chloroisopropyl) phosphate (TCIPP), tri isobutyl phosphate (TIBP), tri-n-butyl phosphate 153
(TNBP), butyl diphenyl phosphate (BdPhP), triphenyl phosphate (TPHP), dibutyl phenyl 154
phosphate (DBPhP), tris(1,3-dichloro-2-propyl)phosphate (TDCIPP), tris(2- 155
butoxyethyl)phosphate (TBOEP), 2-ethylhexyl diphenyl phosphate (EHDP), sum of tricresyl 156
phosphates (sum of TMPP isomers), and tris(2-ethyl hexyl) phosphate (TEHP).
157
2.4.1 POP extraction and clean up 158
Approximately 0.5-1.1 g of plasma was spiked with an internal standard containing labeled 159
standards of PCBs, PBDEs, HCB, chlordane, nonachlor, mirex, HCHs, and DDTs. The 160
plasma samples were subsequently denaturated with ethanol and ammonium sulphate in 161
deionized water. Samples were extracted thrice with n-hexane, and cleaned up on Florisil®
162
(Fisher Scientific, Pittsburgh, USA) solid phase extraction (SPE) cartridges as described by 163
Sandanger et al. (2007).
164
Approximately 500 mg of feathers were washed thoroughly with Milli-Q water and dried 165
overnight at ambient temperature (Jaspers et al. 2007b; Jaspers et al. 2008). Thereafter they 166
were cut into 1 mm pieces, spiked with internal standards (same standard as above), and 167
covered with cyclohexane/acetone 3:1 (v:v) and sonicated for 15 min. Lastly, feather extracts 168
were fractionated with gel permeation chromatography (GPC - Waters Corporation, Milford, 169
Massachusetts, USA) and cleaned up on Florisil® SPE cartridges. Procedures were modified 170
from Dauwe et al. (2005) and Eulaers et al. (2011, 2014).
171
2.4.2 OPE extraction and clean up 172
Due to insufficient sample volume from the remaining individuals, only four individuals (two 173
males and two females) were used in the OPE determination. Approximately 1 mL of plasma 174
was spiked with 20 ng of an internal standard consisting of deuterated D21-TPHP and D27- 175
TNBP (Chiron AS, Trondheim, Norway) before denaturation with acetonitrile and ammonium 176
sulphate in Oasis® HLB cleaned Milli-Q water (Waters Corporation). Samples were 177
centrifuged and the upper acetonitrile phase was transferred to new 15 mL glass centrifuge 178
tubes with 0.5 g of Supelclean™ PSA (primary-secondary amine bonded silica) and 0.2 g 179
magnesium sulphate (Sigma-Aldrich Inc., St. Louis, Missouri, USA). Samples were then 180
Page 9 of 24 centrifuged and supernatant was transferred to new glass tubes and evaporated to 0.2 mL.
181
Lastly, samples were transferred to 2 mL glass vials and 20 ng of deuterated Tris(propyl) 182
phosphate (D21-TPrP) was added as recovery standard (Chiron AS, Trondheim, Norway).
183
Clean up procedures for feather samples were adapted from the protocol described by 184
Eulaers et al. (2014). Briefly, feather samples were washed thoroughly with Milli-Q water and 185
dried overnight at ambient temperature. Hereafter cut into 1mm pieces, spiked with internal 186
standards consisting of deuterated D21-TPHP and D27-TNBP, and incubated for 5 h at 45 °C 187
with hydrogen chloride (HCl, 1 M) and 6 mL of hexane:dichloromethane (4:1; v:v). After 188
liquid/liquid extraction using hexane:dichloromethane (4:1; v:v), extracts were cleaned up on 189
glass SPE columns with primary-secondary amine (PSA) and eluted with methyl tert-butyl- 190
ether (MTBE).
191
2.4.3 Analyte identification and quantification 192
The analysis of PCBs, PBDEs, and OCPs by high-resolution gas chromatography (HRGC) on 193
an Agilent 7890A gas chromatograph equipped with an Agilent 7683B automatic injector and 194
an Agilent 5975C mass spectrometer (Agilent, Folsom, USA), was performed as described by 195
Herzke et al. (2009). Analysis of OPEs using liquid chromatography on a UPLC column (BEH 196
Phenyl, 100 mm x 2.1 mm ID, 1.8 µm particles, Waters Corp., Milford, USA) on an Accella 197
1250 quaternary pump fitted to a Vantage triple quadrupole mass spectrometer was run in the 198
ESI mode (Thermo Fisher Scientific, Waltham, USA). Injections were 10 µL with a mobile 199
phase gradient of 80 % to 0 % of HLB-cleaned Milli-Q water with 0.1 % formic acid and 200
methanol with 0.1 % formic acid and a column flow of 0.3 mL/min to 0.4 mL/min. Limit of 201
detection (LOD) was defined as three times the signal to noise ratio. For validation of results, 202
one blank sample was included for every tenth sample. Four blanks were included in OPE 203
analyses due to very fluctuating background levels. The standard reference material (SRM) 204
used for plasma samples was SRM 1958 human serum from the National Institute of 205
Standards and Technology (NIST), Gaithersburg, Maryland, USA, with an added OPE 206
standard (d21-TPrP) for quality assurance. No SRM was available for feather samples.
207
However, recoveries of the internal standards in feathers were used to assess the analytical 208
Page 10 of 24 quality of the applied method for POPs (65-75%). Recovery of OPEs was from 42 to 128 % 209
with an average recovery of 75 %.
210
2.5 Statistics 211
For statistical analyses, JMP® from SAS Institute Inc., Microsoft Excel® 2013, SigmaPlot 212
13.0, and the free statistical software R (version 3.1.2) (R Core Team, 2015) were used. To 213
investigate the data including compounds with a high percentage of data below LOQ (limit of 214
quantification; LOD times three), we used methods of survival analysis for left-censored data 215
(Gillespie et al. 2010; Helsel 2005, 2006). The distributions of concentrations in feathers and 216
blood were estimated using the reverse Kaplan–Meier (KM) method (Gillespie et al. 2010;
217
Jaspers et al. 2013) for all PBDEs, PCBs, and OCPs where at least one value above the LOD 218
was available. The reverse KM method is non-parametric and presents the distribution 219
without substituting values below LOD (Jaspers et al. 2013). The “survival failure” procedure 220
in JMP 12 (SAS Institute Inc., Cary, NC, USA) was used to estimate the cumulative 221
distribution of each pollutant concentration level. The cumulative distributions can be found in 222
supplementary information. Due to the low number of samples (n=4) for OPEs, they were not 223
included in further statistics. Further statistics on POPs were performed on compounds with 224
more than 50 % of the measurements above LOQ. Levels below LOQ were assigned a value 225
of p × LOQ, were ‘p’ is the proportion of measurements with a value above LOQ (Voorspoels 226
et al., 2002; Jaspers et al., 2007a).
227
The concentrations of the majority of the pollutants were not normally distributed according to 228
the Shapiro-Wilk test of normality. Common logarithmic (base 10) transformations of all POP 229
concentrations were performed in order to approximate normal distribution. Data were 230
checked for homogeneity of variances using Bartlett’s test. ΣPOPs was calculated as the sum 231
of all PCB, PBDE, and OCP levels in each sample (feather and plasma separately).
232
Differences in mean contributions of pollutants to ΣPOPs between colonies were separately 233
investigated for both colonies using one-way ANOVA. A body condition index (BCI) was 234
calculated in order to investigate how the kittiwake body condition correlates with pollutant 235
levels in blood plasma. BCI was expressed as residual mass from the linear regression 236
relating body mass to skull length (r2=0.65, n=19, p<0.001) as described by Chastel et al.
237
Page 11 of 24 (2005). Skull length was used due to its high correlation with body mass (r=0.82, p<0.001).
238
The linear regressions did not vary between sexes(ANCOVA p=0.46).
239
Pearson product-moment coefficients were carried out to evaluate correlations between levels 240
in feathers and blood. Univariate general linear models (GLMs) were performed for ∑OCPs, 241
∑PCBs, ∑PBDEs, and ∑POPs to investigate relations between the pollutant groups, sex, 242
colonies, thyroid hormones, and BCI. Univariate GLMs were performed separately for 243
individual PCB congeners to investigate a possible OH-PCB mediated interference with THs 244
because of their structural resemblance with thyroid hormones. The best models were 245
selected based on stepwise Akaike’s Information Criterion adjusted for low sample sizes 246
(AICc).
247
3. Results 248
The following compounds were detected in plasma in more than 50 % of the 19 samples: p,p’- 249
DDE, HCB, β-HCB, oxy- and trans-chlordane, cis- and trans-nonachlor, mirex, CB -28, -99, - 250
105, -118, -138, -153, -180, -183, -187, and BDE 47. In feathers, p,p’-DDE, HCB, oxy- 251
chlordane, trans-nonachlor, and CB 153 were detected in more than 50 % of the 19 samples.
252
Of the thirteen investigated OPEs, seven were detected in feathers (TCEP, TNBP, TPHP, 253
TBOEP, sum of TMPP isomers, EHDP, and TEHP) and one in plasma (TCIPP).
254
3.1 Levels of pollutants 255
Sexes were pooled since no significant differences were found between sexes for the 256
different pollutant groups in either Blomstrandhalvøya or Krykkjefjellet (p>0.05 in all cases).
257
The mean concentrations of ΣPOPs for Blomstrandhalvøya and Krykkjefjellet were 258
respectively 72.9 ± 8.63 ng/g ww (wet weight) and 29.6 ± 1.67 ng/g ww in plasma, and 13.4 ± 259
3.63 ng/g and 7.08 ± 1.58 ng/g in feathers. The mean concentration of ΣPOPs in plasma for 260
kittiwakes from Blomstrandhalvøya was more than twice as high as the mean concentration of 261
ΣPOPs for kittiwakes breeding in Krykkjefjellet (Fig. 2).
262
No significant differences were found between the colonies in the mean contribution of CB 263
153, -138, -180, and p,p’-DDE to ΣPOPs. These were the major contaminants in plasma for 264
Page 12 of 24 kittiwakes from both Blomstrandhalvøya and Krykkjefjellet constituting 68.5 % and 66.8 % of 265
the total POP load, respectively (figure SI 7 in supplementary information).
266
Pollutant levels in feathers did not differ significantly between colonies for ΣPCBs, ΣPBDEs, 267
and ΣOCPs. CB 153, p,p’-DDE, HCB, OxC, and TN were the only compounds that were 268
detected in more than 50 % of the feather samples, and constituted 33.0 %, 23.0 %, 10.7 %, 269
7.2 %, and 1.8 % of the total POP load in feathers, respectively. The biggest contributor to 270
mean ΣPOPs was ΣPCBs (Blomstrandhalvøya 80.1 % and 51.1 %; Krykkjefjellet 75.7 % and 271
41.8 %, for plasma and feathers, respectively) (Table SI 1).
272
Levels of OPEs were only investigated in four individuals from Krykkjefjellet, since only their 273
sample amounts of plasma and feathers were sufficient for OPE analyses. Two of the feather 274
Figure 2. Comparison of mean concentration of POPs in ng/g ww in plasma ± SE between Blomstrandhalvøya (n=11) and Krykkjefjellet (n=8). Significant differences between the two colonies: *: p<0.05. **: p<0.01. ***: p<0.001.
p,p'-DDE HCB
OxC TC TN CN Mirex
CB 28 CB 99
CB 105 CB 118
CB 138 CB 153
CB 180 CB 183
CB 187 CB 194
BDE 47
C o n c e n tr a ti o n ( n g /g w w )
0 2 4 6 8 10 15 20 25
Blomstrandhalvøya Krykkjefjellet
*
** ***
**
***
**
**
***
***
***
***
***
***
Page 13 of 24
% of total POP load
0 20 40 60 80 100
Plasma Feathers
PCBs PBDEs OCPs
**
**
samples had no detectable levels of any of the investigated OPEs after blank correction. The 275
main contributors to ΣOPEs in the other two feather samples were EHDP and TPHP, with 276
TPHP detected in both feather samples. Only one plasma sample showed OPE levels 277
(TCIPP) above LOQ after blank correction.
278
3.2 Correlations between pollutants in feathers and plasma 279
The mean contribution of ΣPCBs and ΣOCPs to the total contaminant load differed 280
significantly between plasma and feather samples (p=0.002 and p=0.009, respectively) (figure 281
3). Levels of ΣPCBs contributed significantly more to the total contaminant load in plasma, 282
whereas the mean contribution of ΣOCPs in feathers was more than twice as high as in 283
plasma (41.4 % vs 20.4 %, respectively). Pearson correlations between log transformed 284
concentrations of pollutants in plasma and feather samples for the colonies combined 285
revealed no significant correlations, 286
except for a negative relationship for oxy- 287
chlordane (r=-0.58, p=0.008). Due to 288
high differences in plasma contaminant 289
levels between colonies, correlations 290
were also investigated for each colony 291
separately. The only significant 292
relationship between feather and plasma 293
concentrations was for CB 153 in 294
Krykkjefjellet (r=0.81, p=0.02).
295 296
3.3 TH levels 297
fT3 levels differed significantly between sexes (p=0.007), also when body mass was 298
considered (p=0.003), with a range from 2.45 to 6.11 pg/mL for males and 1.25 to 3.48 pg/mL 299
for females. No significant differences in fT3 levels were found between the colonies. TT3 300
levels ranged from 1.68 to 5.12 ng/mL for males and from 0.70 to 3.52 ng/mL for females, but 301
no significant differences were found between sexes nor colonies. The ratio between fT3 and 302
Figure 3. Sum (Σ) of PCBs, PBDEs, and OCPs expressed as mean percentage (%) of total POP load ± SE in plasma and feathers for 19 kittiwakes from Svalbard. **: significant difference between plasma and feather samples, p<0.001.
Page 14 of 24
Figure 4. Correlation plot between TT3:fT3 ratio and the log concentration of CB 187 in 11 kittiwakes from
Blomstrandhalvøya, Kongsfjorden. The p- and r-values are displayed in the upper right corner. The unbroken line is the regression, the red dashed line is the 95 % confidence interval for the regression, and the dotted line is the 95 % confidence interval for the samples.
TT3 ranged from 0.47 to 1.78 for males and from 0.56 to 1.53 for females and did not differ 303
significantly between neither colonies nor sexes.
304
3.4 Associations between contaminants, thyroid hormones, and physiological parameters 305
Body mass of the 19 studied kittiwakes ranged from 300 to 433 g, and an overall significant 306
difference was found between the sexes (p<0.001), with lower body mass in females, as 307
expected. The BCI did not differ significantly between sexes for Krykkjefjellet (p=0.609), but a 308
trend was found for Blomstrandhalvøya (p=0.057), with female kittiwakes from 309
Blomstrandhalvøya having the lowest BCI. Breeding status did not affect body mass or BCI 310
(p=0.25 and p=0.33, respectively). By inspecting GLM regression analyses for the pollutant 311
groups in plasma, the best models comprised BCI and colony for ΣPCBs (F2,16 =25.01, 312
p=0.00001, r2=0.73) and for ΣOCPs (F2,16
313
=8.41, p=0.003, r2=0.45). These findings 314
are supported by GLM regression analyses 315
for ΣPOPs, as the best significant 316
regression analysis comprised both colony 317
and BCI (F2,16 =21.82, p=0.00003, r2=0.70).
318
No significant results were found for 319
explaining the level of ΣPBDEs.
320
CB -28, -138, -187 (all 2, 4, 4’ or 2, 2’, 4 321
substituted), and ΣPBDE were negatively 322
correlated with TT3:fT3 ratio (all r≥-0.60 323
and all p≤0.05) for kittiwakes from 324
Blomstrandhalvøya (CB 187 as example in 325
Fig. 4), but not for Krykkjefjellet. All 326
pollutant groups had a positive, but not 327
significantly, correlation with fT3 levels.
328
Log CB 187 concentration
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8
TT3:fT3
0.0 0.5 1.0 1.5
2.0 r = - 0.67
p = 0.02
Page 15 of 24 4. Discussion
329
4.1 Pollutant levels 330
To the knowledge of the authors, this is the first study to investigate OPEs in feathers and 331
plasma from kittiwakes from Svalbard. Due to elevated levels of OPEs in the blank samples 332
(ranging between 0.03 to 4.47 ng/g ww and 0.02 to 26.5 ng/g feather for plasma and feather 333
blanks, respectively) indicating possible external contamination, most OPE levels in the 334
samples were lower than blank sample concentrations. Therefore, the OPE results should be 335
interpreted with caution. Nevertheless, OPEs show long-range atmospheric transport (OPEs 336
in the Arctic atmospheric are now exceeding both contemporary and historical levels of 337
PBDEs) and bioaccumulative abilities (Salamova et al. 2014). Detections of OPEs in Arctic 338
wildlife are increasing (Hallanger et al. 2015), although most OPEs are readily metabolized 339
(Greaves and Letcher 2014). This warrants a further investigation of OPEs in Arctic wildlife 340
with larger sample sizes.
341
The lack of difference in pollutant load between sexes has been reported previously in liver 342
samples from adult Arctic seabirds as glaucous gulls (Larus hyberboreus; Sagerup et al.
343
2009) and kittiwakes (Buckman et al. 2004; Borgå et al. 2005; Bustnes et al. 2017). Plasma 344
levels of pollutants are variable and highly dependent on the diet (Borgå et al. 2005), and as 345
both female and male kittiwakes nurture nestlings (Coulson 2011), both sexes are supposed 346
to have similar diet and energy expenditure during the feeding period (Barrett et al. 1985).
347
This could partly explain why we found no significant differences in plasma levels of pollutants 348
between sexes.
349
In kittiwakes from the Krykkjefjellet colony, POP levels were lower in feathers but similar in 350
plasma compared to previously reported levels for the same colony (Johnsen 2011; Nordstad 351
et al. 2012; Solheim et al. 2016), independent of sex. However, in kittiwakes from 352
Blomstrandhalvøya, plasma levels of ΣPCBs, HCB, OxC, and p,p’-DDE were more than twice 353
as high than previously reported levels for kittiwakes from Krykkjefjellet (Johnsen 2011;
354
Nordstad et al. 2012). The higher levels of almost all halogenated pollutants found at the 355
Blomstrandhalvøya colony may be caused by several factors. This includes individual 356
Page 16 of 24 variations in breeding status, body size, sex, feeding ecology, and area, which may affect the 357
trophic transfer of pollutants (Henriksen et al. 1996; Borgå et al. 2004). However, similar POP 358
profiles were found for the two colonies, suggesting that their feeding ecology may be similar, 359
but time and energy spent on searching for food may differ.
360
Females from Blomstrandhalvøya were sampled late in the breeding season, and had lower 361
BCI than the rest of the kittiwakes from both colonies. This suggests a higher redistribution of 362
stored lipids, and thereby release of pollutants. As a result, female kittiwakes from 363
Blomstrandhalvøya may experience higher levels of circulating pollutants. Body mass and 364
BCI did not differ significantly between the colonies for male kittiwakes, but males from 365
Blomstrandhalvøya, sampled late in the breeding season still had significantly higher levels of 366
POPs than males from Krykkjefjellet. No differences were, however, found in body condition 367
between breeding and non-breeding kittiwakes in the present study. To further investigate this 368
difference between the two colonies, blood samples from adult breeding female kittiwakes 369
were sampled mid-July 2015 at both colonies. No significant differences between the colonies 370
were found in 2015 (unpublished data, see figure SI 8 in supplementary information), 371
indicating that timing of sampling is of utmost importance when investigating levels and 372
potential effect of POPs in Arctic seabirds.
373
4.2 Correlations 374
In general, only low correlations between feathers and internal levels have previously been 375
reported for aquatic birds (Jaspers et al. 2007a), and correlations between feathers and preen 376
oil have mostly been absent (Solheim et al. 2016).
377
The kittiwake is a migratory bird, and its overwintering areas throughout the North Atlantic 378
differ from its breeding grounds (Strøm 2006; González-Solís et al. 2011; Frederiksen et al.
379
2012). As the sampled primary feathers in kittiwakes grow between September to May, when 380
kittiwakes primarily reside at their overwintering areas (Baird 1994; González-Solís et al.
381
2011), they will not reflect contamination at the Arctic breeding grounds, as opposed to 382
plasma, since most of the kittiwakes do not arrive at Kongsfjorden, Svalbard before April 383
Page 17 of 24 (Strøm 2006). This is illustrated by the different PCB and OCP composition in the reported 384
plasma and feather samples.
385
Although feathers have proven to be good biomarkers for pollution in terrestrial and resident 386
bird species (Dauwe et al. 2005; Jaspers et al. 2007a) kittiwakes are not resident, and 387
feathers sampled from adult migratory birds may not be a good biomarker for pollution at the 388
breeding grounds. Nestling feathers, grown at the breeding ground, would presumably act as 389
better biomarkers for pollution levels. It is important to take these considerations into account 390
to improve future studies on migratory marine bird species, like the kittiwake.
391
4.3 Thyroid hormones and pollution 392
Plasma levels of TT3 were similar to previously reported TT3 levels in kittiwakes (Rønning et 393
al. 2008; Johnsen 2011). However, mean fT3 were lower than previously reported levels for 394
both male and female kittiwakes (Welcker et al. 2013). Rønning et al. (2008), Johnsen (2011), 395
and Welcker et al. (2013) all determined fT3 levels by radioimmunoassay (RIA), whereas the 396
current study used an enzyme-linked immunosorbent assay (ELISA). Maybe the use of 397
different assays could explain the reported difference in fT3 levels, although TT3 levels 398
reported were found similar. Male kittiwakes had significantly higher levels of fT3 than 399
females in the current study. Similar results for kittiwakes have been reported (Welcker et al.
400
2013) although these were not significant. Further, in a study by Verreault et al. (2004), 401
reported levels of fT3 in male glaucous gull were 28 % higher than in females. The latter 402
study also found decreasing levels of T4 and T4:T3 ratio with increasing pollutant load, but 403
only for male glaucous gulls, indicating a sex-specific thyrotoxicity. The ratios between THs 404
have previously been described as sensitive indicators of revealing contaminant exposure 405
(Peakall, 1992).
406
No sex differences were found in kittiwakes from Blomstrandhalvøya, yet overall they had 407
significantly higher levels of pollutants than kittiwakes from Krykkjefjellet. Higher levels of 408
circulating contaminants were associated with lower TT3:fT3 levels in Blomstrandhalvøya 409
kittiwakes. As most of the pollutants had a positive, but not statistically significant, correlation 410
with fT3 levels, increased levels of fT3 might be a possible explanation for the decreased 411
Page 18 of 24 TT3:fT3 ratio. Positive correlations between fT3 levels and pollutant levels have previously 412
been reported in glaucous gulls (Verreault et al. 2004). It has been speculated that 413
thyrotoxicity is sex-specific, but both males and females have been reported as seemingly 414
more susceptible to thyrotoxicity (Verreault et al. 2004; Melnes et al. 2017). The positive 415
correlations between fT3 levels and pollutant levels reported in the current study, although not 416
significant, might partly explain the significantly higher levels of fT3 found in males from both 417
colonies. Pollutant mediated interference with TH plasma carrier proteins has been 418
suggested, as some OH-PCBs have structural resemblance with THs (Verreault et al. 2004).
419
As avian transthyretin has higher affinity for T3 than T4 (Chang et al. 1999), it is possible that 420
most transthyretin will be saturated with T3. The displacement of T3 from transthyretin by 421
organic contaminants could facilitate excretion of T3, thereby reducing levels of TT3 in 422
plasma and cause the TT3:fT3 ratio to decrease with increasing levels of pollutants (Blévin et 423
al. 2017).
424
The significant correlations reported in the current study may possibly be representing a 425
potential pollutant mediated influence on the thyroid system, as high levels of circulating 426
contaminants were associated with a lower TT3:fT3 ratio. However, adaptive responses to 427
food availability and fasting during the breeding period may also cause a decrease in T3 428
levels, especially in birds (McNabb 2007), resulting in a possible covariation between 429
increasing levels of circulating pollutants and a lower TT3:fT3 ratio. Further studies including 430
a larger sample size, histology, T4 levels, and glandular hormones would be necessary to 431
draw definite conclusions regarding the observed relations.
432
5. Conclusion 433
This study is the first to report detection and quantification of OPEs in kittiwake feathers from 434
Svalbard and emphasize their occurrence in Arctic wildlife. Further studies with a larger 435
sample size are required to conclude on trends and population levels. This study provides 436
new insights into the applicability of using feathers as biomonitors of exposure for emerging 437
and legacy pollutants. Our results suggest low usability of adult kittiwake feathers when 438
investigating contamination at the local breeding colony, in contrast to plasma levels.
439
Therefore, adult migratory bird feathers are not recommended for biomonitoring pollutants at 440
Page 19 of 24 breeding grounds, while nestling feathers, or feathers grown at the breeding grounds, may 441
serve as a more reliable biomonitor. Moreover, the significant correlations found in this study 442
between the BCI, TT3:fT3 ratio and several POPs, warrants further investigation of the 443
observed relations during the breeding season. Our study further underpins that timing of 444
sampling is of utmost importance when investigating levels and potential effects of organic 445
pollutants in Arctic seabirds.
446
Acknowledgments 447
We thank Pierre-Axel Monternier for assistance during fieldwork, the staff of Sverdrup 448
Research Station in Ny-Ålesund for logistic support, and Arntraut Götsch from NILU for 449
support during the chemical analysis of the samples.
450
Compliance with ethical standards 451
N. B. Svendsen and V. L. B. Jaspers received Artic Field Grants (ES520962 and ES520958, 452
respectively) from the Norwegian Research Council to carry out the fieldwork at Svalbard, 453
Norway. V.L.B Jaspers is further supported by the Norwegian University of Science and 454
Technology (NTNU), and the pollutant analyses were funded in a joint collaboration between 455
NTNU and the Norwegian Polar Institute. All applicable international, national, and/or 456
institutional guidelines for the care and use of animals were followed. The sampling from 457
kittiwakes at Svalbard occurred in accordance with approval from the Norwegian Animal 458
Research Authority (FDU permission number 2014/59453-2). The authors confirm that no 459
competing personal or financial interests exist regarding the submitted manuscript.
460
Supplementary information 461
Supplementary information (SI) includes cumulative probability plots for PCBs, PBDEs and 462
OCPs in blood and feathers, levels of OPEs in blood and feathers, and comparisons between 463
POP levels at the two colonies. SI is available online.
464
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